My laboratory is interested in the molecular basis of cellular interactions during development. Our present approach to this problem evolved from biochemical studies of the T/t-complex mutations in the mouse - mutations that interfere, directly or indirectly, with normal fertilization and embryonic morphogenesis. Of nine enzymes that were assayed, only beta 1,4 galactosyltransferase (GalTase) proved to be different between normal and T/t-mutant tissues. Consequently, for the past 13 years, we have used a variety of biochemical, immunological, and genetic probes to define the function of cell surface GalTase participates during the initial binding of sperm to the egg zona pellucida, during the transition of the compacted morula to the blastocyst, and during mesenchymal cell migration on laminin- containing matrices. This award was initiated two years ago to bring the sophistication of molecular biology to study the regulation, expression and function of surface GalTase. During the current funding period we have defined the temporal involvement of surface GalTase in preimplantation embryonic development and F9 embryonal carcinoma (EC) cell adhesion, cloned a 3.1kb GalTase cDNA from F9 EC cells, used this cDNA to clarify the confusion in the literature regarding the isolation of multiple, unrelated GalTase cDNAs, analyzed GalTase expression and subcellular distribution during F9 EC cell differentiation, and cloned the full-length GalTase cDNA from lactating mammary gland libraries. During the next funding period, we intend to continue our studies regarding the regulation of GalTase expression and function during development. Specifically, we will determine if surface GalTase triggers F9 EC cell differentiation, analyze the relationship between the Golgi and plasma membrane forms of GalTase by appropriate GalTase cDNA transfections and subcellular fractionation, analyze GalTase expression during normal and t- mutant spermatogenesis by Northern blot analysis and in situ hybridization, and examine the consequences of altering GalTase expression in transfected cells and in transgenic animals. These studies will offer new insights into the regulation of glycoconjugate biosynthesis in general, and more importantly, address the mechanisms underlying mammalian fertilization and embryonic development at the molecular level. We will attempt to produce genetically-defined phenocopies of the T/t-complex mutations by over producing GalTase in transgenic animals.